title: Expression and purification of dengue virus NS5 polymerase and development of a high-throughput enzymatic assay for screening inhibitors of dengue polymerase.
abstract: The nonstructural protein 5 (NS5) of dengue virus (DENV) plays a central role in the virus replication. It functions as a methyltransferase and an RNA-dependent RNA polymerase. As such, it is a promising target for antiviral drug development. To develop a high-throughput biochemical assay for screening compound libraries, we expressed and purified the polymerase domain of the dengue NS5 protein in bacterial cells. The polymerase activity is measured using a scintillation proximity assay. This homogeneous and high--throughput assay enables screening of compound libraries for identifying polymerase inhibitors against DENV. In this chapter we describe the methods to express and purify the dengue NS5 polymerase from E. coli and a validated high-throughput enzymatic assay for screening inhibitors of NS5 polymerase.
authors.affiliation: Janssen Infectious Diseases-Diagnostics BVBA, Johnson & Johnson Corporation, Beerse, Belgium.
authors.forename: Edwin Yunhao
authors.initials: EY
authors.lastname: Gong
authors.forename: Hannah
authors.initials: H
authors.lastname: Kenens
authors.forename: Tania
authors.initials: T
authors.lastname: Ivens
authors.forename: Koen
authors.initials: K
authors.lastname: Dockx
authors.forename: Katrien
authors.initials: K
authors.lastname: Vermeiren
authors.forename: Geneviève
authors.initials: G
authors.lastname: Vandercruyssen
authors.forename: Benoit
authors.initials: B
authors.lastname: Devogelaere
authors.forename: Pedro
authors.initials: P
authors.lastname: Lory
authors.forename: Guenter
authors.initials: G
authors.lastname: Kraus
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D003716:Dengue Virus / Q000187:drug effects* / Q000502:physiology*; D057075:Enzyme Assays / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D057166:High-Throughput Screening Assays; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014169:Transformation, Bacterial; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; C582223:NS5 protein, dengue virus; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins